Ischemic preconditioning requires opening of pannexin-1/P2X(7) channels not only during preconditioning but again after index ischemia at full reperfusion.

Protection of the ex vivo rat heart from ischemia/reperfusion injury can be provided by ischemic preconditioning (IPC). Previous studies revealed that a complex of pannexin-1 with the P2X₇ receptor forms a channel during IPC that results in the release of cardioprotectants such as adenosine and sphingosine 1-phosphate (S1P) that bind to G-protein-coupled cell surface receptors triggering cardioprotective cell signaling pathways. Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X₇ receptors (brilliant blue G) are known to block IPC when administered at the time of preconditioning (Vessey et al. J Cardiovasc Pharmacol Ther 15:190, 2010). We now demonstrate that these same antagonists also block the cardioprotective effects of IPC when added after the index ischemia during full reperfusion. Likewise, addition at full reperfusion of binding antagonists to the endogenous cardioprotectants S1P (VPC) or adenosine (8-SPT) reduced the effectiveness of IPC. These data suggest that IPC has a component that requires the release of cardioprotectants via pannexin-1/P2X₇ channels not only during preconditioning phase but again during the early stages of reperfusion following the index ischemia. It was found that the level of cardioprotectant release required at reperfusion to achieve cardioprotection was lower when hearts had been preconditioned. Further, pharmacologic preconditioning with S1P or adenosine was also blocked at reperfusion by antagonists of the pannexin-1/P2X₇ channels indicating that pharmacologic preconditioning also requires opening of the channel at full reperfusion. In untreated hearts, key components of the PI3 kinase/Akt signaling pathway were revealed by western analysis to be lost during ischemia. This correlates with an inability to generate phospho-Akt at reperfusion. IPC prevents this loss and thereby primes the cell for response to cardioprotectants released at full reperfusion.